CN103293260B - Method for high-efficiency detection of rhodamine B in food and rapid detection kit - Google Patents
Method for high-efficiency detection of rhodamine B in food and rapid detection kit Download PDFInfo
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- CN103293260B CN103293260B CN201210045581.1A CN201210045581A CN103293260B CN 103293260 B CN103293260 B CN 103293260B CN 201210045581 A CN201210045581 A CN 201210045581A CN 103293260 B CN103293260 B CN 103293260B
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Abstract
The invention relates to a method for high-efficiency detection of rhodamine B in food and a rapid detection kit. The detection method is mainly divided into three steps: 1, extracting rhodamine B in food; 2, purifying and enriching the rhodamine B in food; and 3, detecting through a machine. The corresponding kit consists of an extracting agent 1, an extracting agent 2 and a solid extraction column 3. When the rhodamine B in food is detected by employing the method, the detection limit can reach 0.5ppb, and the external standard recovery rate is over 85 percent. The food is subjected to pretreatment by employing the method and the key reagents such as the extracting agent 1 and the extracting agent 2, so that the rhodamine B in food is completely extracted, the toxicity of the used reagent is obviously reduced, the required time for detecting the rhodamine B in food is greatly shortened, the detection precision is improved, and the method has the excellent characteristics of high convenience, rapidness, environmental friendliness and high applicability and implementability, and a toxic agent is avoided.
Description
Technical field
The present invention is specifically related to rhodamine B efficient detection method and quick detection kit in a kind of food.
Background technology
Rhodamine B, also claims rose red b, is commonly called as pollen red, is a kind of alkaline fluorescent dye.It has been widely used in the fields such as mining industry, environmental protection and iron and steel as fluorescent reagent, be the conventional reagent of fluorescence analysis, and still, in some food, tested finding contained rhdamine B.Due to, it has potential carcinogenic and mutagenicity, and the states such as China and European Union do not allow to add and use in food.Therefore the detection of, food being carried out to rhodamine B is and is important and imperative.
To the detection of rhodamine B in food, mostly adopt high performance liquid chromatography, Liquid Chromatography-Tandem Mass Spectrometry method and vapor-phase chromatography, but also there is no more efficiently so far method report fast.The inspection and quarantining for import/export industry standard " detection method of rhodamine B in import and export food " that China promulgated in 2010, adopt exactly liquid chromatographic detection and liquid chromatography-mass spectrography/Mass Spectrometer Method, the method fully demonstrates the advantage of LC-MS technology, but, because it is in practical operation, the method of pre-treatment is too loaded down with trivial details, the use that particularly GPC purifies, not only increase the workload detecting, and efficiency is also not bery remarkable, also has the use of organic solvent, as cyclohexane, not only poisonous but also harmful, a large amount of uses can have a strong impact on environment, be unfavorable for environmental protection, this kind of method is because of the reason of himself, its application a large amount of in testing agency is restricted.Recently, also have additive method, have report enzyme linked immunosorbent assay to can be used to detect the rhodamine B in food, although this kind of method can detect the rhodamine B in food in a large number, but its accuracy and sensitivity are all not as good as high performance liquid chromatography, Liquid Chromatography-Tandem Mass Spectrometry method and vapor-phase chromatography.
Summary of the invention
The object of the present invention is to provide one simple to operate, rhodamine B efficient detection method and quick detection kit in effective food.
First the present invention provides the quick detection kit of rhodamine B in a kind of food, comprising:
(1) extraction agent 1: be made up of acetonitrile, acetic acid and ammonium formate, wherein the shared volume ratio of acetonitrile is 90-99%; Acetic acid and ammonium formate volume ratio are 1:1;
(2) extraction agent 2: formed by the mass ratio of 1:1:1 by neutral alumina, acidic alumina, alkali alumina;
(3) solid-phase extraction column: the filler of solid-phase extraction column is prepared by the following method: by filler PEP, PSA and C18, the ratio of 2:2:1 is mixed filling post in mass ratio.
The present invention also provides rhodamine B efficient detection method in a kind of food, utilizes above-mentioned quick detection kit to carry out following steps detection to food samples to be measured:
1) get 5 g samples in 50 mL tool plug plastic centrifuge tubes, add 20 mL extraction agents 1, with 8000 rpm homogeneous 2 min, add 5-10 g extraction agent 2, vibration 1 min, centrifugal 10 min of 4500 rpm;
2) get 5 mL supernatants, add 10 mL water dilutions, be prepared into 15 mL sample liquid;
3) 15 mL sample liquid are crossed to solid-phase extraction column, vacuumize, the acetonitrile solution drip washing that is 20% by 5 mL volume fractions;
4) 2 mL are containing 1-5%(v/v) the acetonitrile solution wash-out of glacial acetic acid, eluent adds 0.5 mL 0.1mol/L ammonium acetate solution, is settled to 2.5 mL;
5) filter, upper machine examination is tested, and adopts high performance liquid chromatography or HPLC-MS technology for detection.
The filler of described solid-phase extraction column is prepared by the following method: filler PEP, PSA and C18 are mixed by the mass ratio of 2:2:1, fill with pillar 60-200 mg; The particle mean size 50 μ m of filler, average pore size 60 dusts.
The condition of described high performance liquid chromatography is chromatographic column: C18 post, granularity 5 μ m; Mobile phase: acetonitrile: 5mmol/L ammonium acetate solution by volume=75:25; Flow velocity, 0.2 mL/min; Column temperature, 35 DEG C; Sample size, 10 μ L.
Wherein, the use of combining of extraction agent 1 and extraction agent 2 is extracted the rhodamine B in food, the use of solid-phase extraction column as much as possible, not only having reduced sample liquid suppresses the ion of machine, extract and reclaim but also greatly improved, use this pillar, can make the recovery be stabilized in more than 85%.
The extraction agent 1 the present invention relates to and the use of extraction agent 2, be not limited to the quantitative amounts that the present invention provides, as long as according to this proportioning, can be prepared into different product forms; Solid-phase extraction column is also not limited to the quantity of the perfusion filler relating in the present invention.Can be according to the use of extraction agent 1 and extraction agent 2, coordinate and change the specification of pillar and the quantity of filler.
The rhodamine B quick detection kit the present invention relates to comprises extraction agent 1, extraction agent 2 and solid-phase extraction column, sample is processed, adopt the efficient detection method of this kit, the content of rhodamine B in can fast detecting food, includes but not limited to the mensuration of rhodamine B in cured fish, bacon, sausage, fruit juice, jam, onion, candy, preserved plum, chilli powder and biscuit etc.Wherein, the data after mensuration are calculated with the following method:
Wherein:
The content of rhodamine B in X test sample, μ g/kg;
N test sample extension rate;
The concentration of rhodamine B in the sample obtaining in C typical curve, ng/nL;
C
0the concentration of rhodamine B in the blank test obtaining in typical curve, ng/nL;
The final constant volume of V sample solution, mL;
The quality of M test specimen, g.
Apply the content that this kind of method detects rhodamine B in sample, lowest detection line can reach 0.5 μ g/kg, exceedes existing other conventional detection methods and approaches 10 times, and it is significant as can be seen here.
The present invention is to provide rhodamine B efficient detection method and quick detection kit in a kind of food, set up the pre-treating method of rhodamine B in brand-new efficient detection food, corresponding kit has been simplified the step of experiment greatly, as long as extraction agent 1, the use that extraction agent 2 and Solid-Phase Extraction are lived, just pre-treatment is simplified to 3 steps, to follow-up high performance liquid chromatography, Liquid Chromatography-Tandem Mass Spectrometry method is processed the detection of rear sample, also make its experimental procedure easier, fast, and avoid applying the poisonous and harmful reagent such as cyclohexane, not only save testing cost, also greatly improved the accuracy detecting, sensitivity and more environmental protection.
Brief description of the drawings
Fig. 1 is that rhodamine B standard items LC-MS detects collection of illustrative plates;
In Fig. 2 chilli powder 1# sample, the LC-MS of mark-on rhodamine B detects collection of illustrative plates;
In the cured fish 2# of Fig. 3 sample, the LC-MS of mark-on rhodamine B detects collection of illustrative plates;
In Fig. 4 jam 3# sample, the LC-MS of mark-on rhodamine B detects collection of illustrative plates.
Embodiment
Below in conjunction with representational embodiment, further set forth the present invention.These implementation examples are only not used in and limit the scope of the invention for the present invention is described.The related detection of following all embodiment of enumerating all has the method for quick the present invention relates to and is not limited to cited function.
Embodiment 1:
Accurately take chilli powder 5 g that smash to pieces, pack in the plastic centrifuge tube of 50 mL tool plugs, adopt outer adding method to add rhodamine B, concentration is that 14.4 ng/mL(are the rhodamine B of adding 14.4 ng in every milliliter of extract 1), then add 20ml extraction agent 1 of the present invention, with 8000 rpm homogeneous 2 min, add 5g extraction agent 2,1 min that vibrates, centrifugal 10 min of 4500 rpm; Accurately get 5 mL supernatants with pipettor, with 10 mL distilled water dilutings, be prepared into 15 mL sample liquid, for subsequent use; 15 mL sample liquid are crossed to solid-phase extraction column (LHSPE), vacuumize, 5 mL 20% acetonitrile/water solution drip washing; 2 mL 2% glacial acetic acid/acetonitrile wash-outs, add 0.5 mL 0.1mol/L ammonium acetate solution, are settled to 2.5 mL; Employing filtering membrane filters, and adopts high performance liquid chromatography or HPLC-MS (LC-MS) technology for detection, and upper machine examination is tested, chromatographic condition: C18 post, 150 mm × 2.1 mm(internal diameters), granularity 5 μ m; Mobile phase, acetonitrile+5 mmol/L ammonium acetates (75+25, V/V); Flow velocity, 0.2 mL/min; Column temperature, 35 DEG C; Sample size, 10 μ L.Obtain data collection of illustrative plates as Fig. 2.Fig. 1 is the data collection of illustrative plates of the rhodamine B standard items of same concentration.According to the method for the conventional peak area ratio of external standard method, the recovery that obtains rhodamine B in this sample chilli powder sample number into spectrum 1# is 88.5%.
This experiment repeats 4 times, applies the same solid-phase extraction column the present invention relates to, and obtains the same recovery, and post effect is constant.
The kit adopting is as follows:
(1) extraction agent 1: be made up of acetonitrile, acetic acid and ammonium formate, wherein the shared volume ratio of acetonitrile is 90%; It is 5%, 5% that acetic acid and ammonium formate respectively account for volume ratio;
(2) extraction agent 2: formed by the mass ratio of 1:1:1 by neutral alumina, acidic alumina, alkali alumina;
(3) solid-phase extraction column: the filler of solid-phase extraction column is prepared by the following method: by filler PEP, PSA and C18, the ratio of 2:2:1 is mixed filling post in mass ratio.Fill with pillar 60 mg; The particle mean size 50 μ m of filler, average pore size 60 dusts.
Embodiment 2:
Accurately take cured fish 5 g that smash to pieces, pack in the plastic centrifuge tube of 50 mL with stopper, adopt outer adding method to add rhodamine B, concentration is 14.4 ng/mL, then add 20ml extraction agent 1 of the present invention, with 8000 rpm homogeneous 2 min, add 10g extraction agent 2,1 min that vibrates, centrifugal 10 min of 4500 rpm; Accurately get 5 mL supernatants with pipettor, with 10 mL distilled water dilutings, be prepared into 15 mL sample liquid, for subsequent use; 15 mL sample liquid are crossed to solid-phase extraction column (LHSPE), vacuumize, 5 mL 20% acetonitrile/water solution drip washing; 2 mL 2% glacial acetic acid/acetonitrile wash-outs, add 0.5 mL 0.1mol/L ammonium acetate solution, are settled to 2.5 mL; Employing filtering membrane filters, and adopts high performance liquid chromatography or HPLC-MS (LC-MS) technology for detection, and upper machine examination is tested, chromatographic condition: C18 post, 150 mm × 2.1 mm(internal diameters), granularity 5 μ m; Mobile phase, acetonitrile+5 mmol/L ammonium acetates (75+25, V/V); Flow velocity, 0.2 mL/min; Column temperature, 35 DEG C; Sample size, 10 μ L.Obtain data collection of illustrative plates as Fig. 3.Fig. 1 is the data collection of illustrative plates of the rhodamine B standard items of same concentration.According to the method for the conventional peak area ratio of external standard method, the recovery that obtains rhodamine B in the cured fish sample number into spectrum of this sample 2# is 90.2%.
This experiment repeats 4 times, applies the same solid-phase extraction column the present invention relates to, and obtains the same recovery, and post effect is constant.
(1) extraction agent 1: be made up of acetonitrile, acetic acid and ammonium formate, wherein the shared volume ratio of acetonitrile is 99%; Acetic acid and ammonium formate volume ratio are 1:1;
(2) extraction agent 2: formed by the mass ratio of 1:1:1 by neutral alumina, acidic alumina, alkali alumina;
(3) solid-phase extraction column: the filler of solid-phase extraction column is prepared by the following method: by filler PEP, PSA and C18, the ratio of 2:2:1 is mixed filling post in mass ratio.Fill with pillar 100 mg; The particle mean size 50 μ m of filler, average pore size 60 dusts.
Embodiment 3:
Accurately take strawberry jam 5 g, pack in the plastic centrifuge tube of 50 mL with stopper, adopt outer adding method to add rhodamine B, concentration is 14.4 ng/mL, then add 20ml extraction agent 1 of the present invention, with 8000 rpm homogeneous 2 min, add 7g extraction agent 2,1 min that vibrates, centrifugal 10 min of 4500 rpm; Accurately get 5 mL supernatants with pipettor, with 10 mL distilled water dilutings, be prepared into 15 mL sample liquid, for subsequent use; 15 mL sample liquid are crossed to solid-phase extraction column (LHSPE), vacuumize, 5 mL 20% acetonitrile/water solution drip washing; 2 mL 2% glacial acetic acid/acetonitrile wash-outs, add 0.5 mL 0.1mol/L ammonium acetate solution, are settled to 2.5 mL; Employing filtering membrane filters, and adopts high performance liquid chromatography or HPLC-MS (LC-MS) technology for detection, and upper machine examination is tested, chromatographic condition: C18 post, 150 mm × 2.1 mm(internal diameters), granularity 5 μ m; Mobile phase, acetonitrile+5 mmol/L ammonium acetates (75+25, V/V); Flow velocity, 0.2 mL/min; Column temperature, 35 DEG C; Sample size, 10 μ L.Obtain data collection of illustrative plates as Fig. 4.Fig. 1 is the data collection of illustrative plates of the rhodamine B standard items of same concentration.According to the method for the conventional peak area ratio of external standard method, the recovery that obtains rhodamine B 3 in this sample strawberry jam sample number into spectrum 3# is 86.1%.
This experiment repeats 4 times, applies the same solid-phase extraction column the present invention relates to, and obtains the same recovery, and post effect is constant.
(1) extraction agent 1: be made up of acetonitrile, acetic acid and ammonium formate, wherein the shared volume ratio of acetonitrile is 95%; Acetic acid and ammonium formate volume ratio are 1:1;
(2) extraction agent 2: formed by the mass ratio of 1:1:1 by neutral alumina, acidic alumina, alkali alumina;
(3) solid-phase extraction column: the filler of solid-phase extraction column is prepared by the following method: by filler PEP, PSA and C18, the ratio of 2:2:1 is mixed filling post in mass ratio.Fill with pillar 200 mg; The particle mean size 50 μ m of filler, average pore size 60 dusts.
Claims (4)
1. a quick detection kit for rhodamine B in food, comprising:
(1) extraction agent 1: be made up of acetonitrile, acetic acid and ammonium formate, wherein the shared volume ratio of acetonitrile is 90-99%; Acetic acid and ammonium formate volume ratio are 1:1;
(2) extraction agent 2: formed by the mass ratio of 1:1:1 by neutral alumina, acidic alumina, alkali alumina;
(3) solid-phase extraction column: the filler of solid-phase extraction column is prepared by the following method: by filler PEP, PSA and C18, the ratio of 2:2:1 is mixed filling post in mass ratio.
2. a rhodamine B efficient detection method in food, is characterized in that: utilize the quick detection kit described in claim 1 to carry out following steps detection to food samples to be measured:
1) get 5 g samples in 50 mL tool plug plastic centrifuge tubes, add 20 mL extraction agents 1, with 8000 rpm homogeneous 2 min, add 5-10 g extraction agent 2, vibration 1 min, centrifugal 10 min of 4500 rpm;
2) get 5 mL supernatants, add 10 mL water dilutions, be prepared into 15 mL sample liquid;
3) 15 mL sample liquid are crossed to solid-phase extraction column, vacuumize, the acetonitrile solution drip washing that is 20% by 5 mL volume fractions;
4) 2 mL are containing the acetonitrile solution wash-out of 1-5%v/v glacial acetic acid, and eluent adds 0.5 mL 0.1mol/L ammonium acetate solution, is settled to 2.5 mL;
5) filter, upper machine examination is tested, and adopts high performance liquid chromatography or HPLC-MS technology for detection.
3. rhodamine B efficient detection method in a kind of food according to claim 2, is characterized in that: the filler of described solid-phase extraction column is prepared by the following method: filler PEP, PSA and C18 are mixed by the mass ratio of 2:2:1, fill with pillar 60-200 mg; The particle mean size 50 μ m of filler, average pore size 60 dusts.
4. rhodamine B efficient detection method in a kind of food according to claim 2, is characterized in that: the condition of described high performance liquid chromatography is chromatographic column: C18 post, granularity 5 μ m; Mobile phase: acetonitrile: 5mmol/L ammonium acetate solution by volume=75:25; Flow velocity, 0.2 mL/min; Column temperature, 35 DEG C; Sample size, 10 μ L.
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| CN105353025A (en) * | 2015-06-19 | 2016-02-24 | 南京工业大学 | Method for rapid determination of rhodamine B in food |
| CN106290673B (en) * | 2016-09-27 | 2018-05-18 | 深圳市计量质量检测研究院 | A kind of method that eutectic solvent extraction liquid chromatography quickly measures rhodamine B |
| CN108459118A (en) * | 2017-12-12 | 2018-08-28 | 铜陵市天屏山调味品厂 | The preprocess method that rhodamine B measures in a kind of flavouring |
| CN109541088A (en) * | 2018-11-28 | 2019-03-29 | 北京农业质量标准与检测技术研究中心 | A kind of purification method of patulin, solid-phase extraction column and its application |
| CN110940762B (en) * | 2019-12-06 | 2022-07-22 | 嘉峪关市食品药品和医疗器械检验检测中心 | Preparation method and application of solid-phase extraction filler and solid-phase extraction column for basic dye in food |
| CN112730644A (en) * | 2020-12-04 | 2021-04-30 | 广东省核工业地质局辐射环境监测中心(广东省铀资源储量评审中心) | Method for determining rhodamine B in chili oil |
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